The overall goal of the procedure is to use robotics and standardized assays to streamline isolation of temperature-sensitive lethal mutations in Chlamydomonas in order to determine essential genes and pathways in this organism. We're interested in both conservation and divergence of essential cellular functions in eukaryotes. And the way we like to study that is with mutations that disrupt essential genes in these processes.
In order for that to work well, you need a really comprehensive collection of mutants and the methods you'll hear about are our way of generating such a collection. We're working in the green algae Chlamydomonas reinhardtii as a representative of the plant kingdom. The main advantage of this technique is the temperature-sensitive lethal mutations can likely be found in every essential pathway.
The method requires no prior knowledge or targeted mutagenesis. Molecular function of the mutated gene suggested immediately from the lethal phenotype. This helps us select interesting mutations disrupting diverse siala pathways beyond cell cycle control.
To carry out UV mutagenesis culture Chlamydomonas cells up to an OD 750 of 0.2 to 0.5 in 100 milliliters of Tris-Acetate-Phosphate, or TAP, under light at 25 degrees Celsius and shaking at 100 RPM. UV mutagenesis is performed independently in two genetic backgrounds according to the text protocol. Check a sample of each culture under the microscope to ensure that the cells are viable and without contamination.
Next dilute the culture to an OD 750 of 0.003 then wrap the bottle with aluminum foil to ensure homogeneous density as the strain is modal and swims directionally in response to light. After adjusting the density of the suspension according to the text protocol, attach a small tube cassette that fits a liquid dispenser and perform a series of washes for sterilization, according to the manufacture's instructions, to prevent contamination. Using an eight syringes liquid dispenser cassette, dispense four by 96 drops of two microliters each of culture onto dry rectangular plates.
Tap gently at the edge of the plate to ensure the merging of all drops into a thin sheet of liquid and immediately cover the plates to prevent exposure to light. When dried, place the plates under a germicidal UV lamp for periods of time determined empirically to give an optimal yield of ts mutants among the survivors. Next, transfer the plates into the dark for eight to 24 hours at room temperature.
Then place the plates in a 21 degree Celsius incubator with illumination. Following approximately 10 days when colonies are grown but not merged, load the plates in the relevant stack as sources for robotic colony picking. Then pick colonies for 384 arrays on rectangular plates and grow them at 21 degrees Celsius with illumination for approximately one week.
Using a replica plating robot, condense the 384 arrays into a 1536 array and allow the plates to grow in the 21 degree Celsius incubator for approximately three days. Replicate the 1536 arrays to two plates each and place one in the 21 degree Celsius incubator and the other in a 33 degree Celsius incubator. Following 24 hours at 33 degrees Celsius, replicate the plates from the 33 degree Celsius incubator to a new set of pre-warmed plates and place them in the 33 degree Celsius incubator.
Following three days of growth at 33 degrees Celsius and five days of growth at 21 degrees Celsius, use a digital camera to photograph a grid plate marked with nine alignment indicators. Then photograph the plates, alternating 21 degree Celsius plates followed with the corresponding 33 degree Celsius plates with all plates placed in a fixed frame to preserve exact orientation. Process the paired 21, 33 plate images with a custom matte lab image analysis software to eliminate the background and to segment the images into 1536 array.
The program will determine the detected bio-mass as total pixel intensity in each position. Load the list of selected colonies generated by the software as an instruction file for the single colony picking robotics. Then prepare the source and target plates according to the robotics instructions and allow the robot to pick the selected colonies to an array.
Place the target plates in the 21 degree Celsius incubator for approximately five days to grow a stock plate. After carrying out a second bio-mass accumulation assay and replicating 100 block plates according to the text protocol, replicate the fresh copy of the 100 block plates to three copies. Following the setup of the third plate in the robot, and spotting of the colonies, take photo-micrographs of a region of each spot of the screening plates at times zero and place the plates at 33 degrees Celsius for incubation.
At varying time points after removing the screening plates from the 33 degree Celsius incubator, quickly take photo-micrographs, making sure the plate holder and the stage controller are precisely calibrated to get images of the same cells in every time point. Analyze microscopic images and select for mutants based on the desired criteria. Spot the final selected set in a 96 arrayed agar plate, ensuring that each plate contains mutants of the same mating type and drug resistance.
Transfer large amounts of the arrayed colonies to Nitrogen free gamete induction medium on 96 well microplates. Incubate the plates under light for approximately five hours to allow gametogenesis. Suspend queries with the opposite mating types harboring the alternative resistance cassettes into tubes with Nitrogen free gamete induction medium for gametogenesis.
Mix the samples from a target plate in a mating mixture volume of 20 microliters. After approximately 10 minutes under the light, spot five microliters from each well twice. Once on a TAP plate for linkage testing and once on a TAP plus five micromole paro plus nine micromole hygro for complementation testing.
After incubating the complementation testing plates, replicate the plates into two copies for ts phenotype identification. Test colonies for ts phenotype according to the text protocol. After eradiation of single cells of Chlamydomonas, cells are allowed to grow for ten days at a permissive temperature, then picked in an arrayed format, as seen here.
The resulting plates in the 384 format are merged into a 1536 array. Three UV exposure times were tested to generate Chlamydomonas ts mutants. Empirically the 1.5 minute exposure time yielded the most ts mutants.
However, by far, the one minute exposure time yielded most of the cell cycle candidates. In this experiment, two sequential ts phenotype assays were performed and around 3000 ts mutants were isolated and phenotypically characterized by time-lapsed microscopy. As seen here, to remove highly recurrent genes from the down-streamed pipeline, complementation and linkage testing with already characterized genes with more than two alleles, were performed against newly collected candidates.
These colonies show no complementation with the query and are therefore a new ts alleles for the queried gene. These are generally excluded from further characterization. Once mastered this technique can be done in high through-put.
Thousands of ts mutants can be isolated in only two or three mutagenesis rounds. A key step after isolation of the ts mutants is to select a subset for further study and it's very interesting to see the range of lethal phenotypes. Phenotyping provides hints to molecular function and also helps us to prioritize mutants for further study.
Keep in mind that the mutants can have hundreds or thousands of mutations in their genomes. Usually only one is causative, though sometimes two mutations are required for the ts phenotype and this can be determined by tetra analysis. Following this procedure, causative mutations can be identified by next generation sequencing analysis of pooled The identified genes will sometimes have mutations suggesting function or they can be new, unknown sequences, which is interesting and exciting.
Chlamydomonas has been a terrific model system for studying cell biology in the plant kingdom. The procedures you heard about should open up study of this organism for essential processes which have been relatively unexamined and we hope that the results will also be relevant to the broader plant kingdom as well.
